Particles, beads and microcapsules have many uses in many areas of technology. The formation of particles or microcapsules can occur using various reactions at a molecular level, such as interfacial polymerization procedures, complex coacervation procedures, gelation (thermal or ionic), precipitation, and so forth. Regardless of the details at a molecular level, however, the process always depends on the prior formation of droplets of suitable dimensions which can then be converted to solid or semisolid form by the above mechanisms.
For example, a preparation containing microparticles encapsulating oil soluble vitamins can be prepared by dropwise extrusion, into an aqueous calcium chloride solution, of an emulsion that consists of the oil phase dispersed in an aqueous sodium or potassium alginate solution. The liquid droplets so formed are rapidly converted to calcium alginate beads by ionic gelation when they fall into a CaCl.sub.2 solution. These beads entrap and retain dispersed oil droplets. The alginate gelation procedure outlined above is not limited to immobilizing oil droplets. One can use this procedure to immobilize live plant and animal cells, bacteria, fungi, nematodes, a range of parasites, active enzymes, and intact organelles like islets of Langerhans and plant chloroplasts.
C. Thies and F. Linek, U.S. Pat. No. 4,464,317, disclosed a method whereby soluble silicate solutions containing active agents are extruded dropwise into aqueous calcium chloride solutions, thereby forming solid silicate beads or capsules loaded with active agent.
Microcapsules can also be formed by interfacial polymerization. In this technology, the material to be encapsulated is dissolved in a liquid (liquid 1) (e.g., an oil). A reactive agent is then also dissolved or suspended in liquid 1. Said reactive agent can be an acid chloride, isocyanate, epoxide, aldehyde, etc. The resulting mixture is extruded dropwise into a liquid (liquid 2) with which it is immiscible. Liquid 2 contains a coreactant for the reactant carried by liquid 1 (e.g., amines, hydroxy groups, etc.). The two coreactants meet at the liquid/liquid interface to thereby form a capsule. This polymerization process assumes the coreactants in liquids 1 and 2 are multifunctional. The encapsulation process can be carried out when liquid 1 is a water-immiscible liquid and liquid 2 is water. Liquid 2 could also be any other liquid immiscible with liquid 1. The process can also be reversed (i.e., liquid 1 is water or water-miscible and liquid 2 is water-immiscible).
The above-mentioned process can also be carried out by dropping an aqueous solution that contains reagent 1 into a second aqueous solution that contains reagent 2. The two reagents react at their mutual interface to form a capsule, as described by Pommerening, K., et al, Biomed Biochem Actu (1983) 42:813. Cell-loaded capsules are formed by ejecting a dispersion of cells in aqueous cellulose sulfate dropwise into a 2% aqueous solution of poly(dimethyl diallyl ammonium chloride). These two polyelectrolytes interact spontaneously to form a hydrogel capsule membrane.
In addition, particles can be formed by dropwise extrusion of a solution that gels when cooled or heated, where the solution is loaded with any active agent or other material desired. For example, when an aqueous gelatin solution is dispersed into cold, water-immiscible solvents, the gelatin solution forms gel particles. Madan, P. L., et al, J Pharm Sci (1978) 67:409-411, prepared spherical gelatin beads loaded with sodium salicylate solution into cold (5.degree. C.) USP mineral oil. The product was dehydrated by acetone washing and isolated as a free-flow powder. Other materials which solidify on cooling include agar, pectin, and carrageenan. Emulsions of an oil or suspension of solid particles in solutions of these materials are encapsulated by dropwise extrusion into cold solvents.
Nakoma, M., et al, J Pharm Pharmacol (1979) 31:869-872, outline a procedure whereby a drug, sulfamethazole, was entrapped in an aqueous agar gel bead by thermal gelation. The beads were formed by dropwise extrusion of the aqueous drug/alginate suspension into a series of cold, water-immiscible, solvents. Beads isolated by gelation in a water-immiscible solvent were irregular (oval or coin) shaped.
Microcapsules can also be formed by complex coacervation. In this technology, originally disclosed by Green, B. K., and Schleicher, L., U.S. Pat. No. 2,800,457, gelatin and any combination of many polyanions (e.g., gum arabic, sodium alginate, carrageenan, etc.) are combined at warm temperatures (e.g., above 35.degree. C.) to form a liquid coacervate. This liquid coacervate is in equilibrium with a dilute polymer solution called the supernatant. The two phases can be readily separated. If a water-insoluble liquid or solid is dispersed in the warm coacervate, the resulting mixture can be extruded dropwise into chilled (5.degree. C.) supernatant phase to thereby produce gelled microcapsules. The capsules so formed can then be cross-linked or fixed by reaction with aldehydes like glutaraldehyde or with tannic acid.
Polymer precipitation is another approach to the formation of microparticles or capsules in which a dispersion or solution of active agent in a polymer/solvent solution is extruded into a nonsolvent for these materials. As an illustration of this type of process, Madan, P. L., et al, J Pharm Pharmac (1978) 30:65-67, extruded dropwise an aqueous solution of cellulose acetate phthalate and sodium salicylate into 5M aqueous HCl. The freshly formed capsules were uniform spheres. However, after drying, they ceased to be perfect spheres because of solvent loss due to evaporation. The dried particles did not return to spherical shape or increase in size when hydrated.
Still another approach to microparticles is described as the "hot melt" process. In this process, an active agent or a solution of active agent is emulsified or dispersed in a molten fat, wax, polymer or mixture of these ingredients. The mixture is then extruded dropwise into a cooling bath immiscible with the melt to form microcapsules or microparticles.
A different process is described in U.S. Pat. No. 4,352,883 issued to F. Lim and R. D. Moss. According to this patent, live cells or tissue are trapped in a calcium alginate gel by dropwise extrusion of a suspension of live cells or tissue in sodium or potassium alginate into dilute calcium chloride that is isotonic in nature and buffered to pH 7.2. The calcium alginate beads are then treated sequentially with a polycation and mild chelating agent (e.g., sodium citrate) to thereby yield macrocapsules with a semipermeable hydrogel membrane that encloses a liquid core that contains live cells or tissue. These capsules protect the cells and tissue from the external environment. Such capsules can be used for a variety of purposes including cell culture, drug release, chromatography support, etc.
Because the formation of microcapsules is so useful in the growth of assorted plant/animal, e.g., mammalian cells, Bellco Technology (Vineland, N.J.) markets a large bioreactor (several-liter capacity) which includes a multitip pipetting device that consists of a number of syringe needles through which cell- or tissue-loaded alginate droplets are extruded into calcium chloride solution to form calcium alginate beads. Significantly, this multitip pipetting device has no gas stream passing over the needle tips and hence has no way to control droplet (and, hence, ultimate particle size) other than by altering the size of the extrusion needle. It is not possible to produce gel beads that are less than 1.5 to 2.0 mm by this method. This bead size is too large to permit or encourage growth of live cells in the interior of the beads due to limitations of oxygen and nutrient diffusion into the interior of the beads. This is a major design limitation of the Bellco Apparatus. In addition, the Bellco apparatus is not designed to extrude small amounts of material. It is a costly and complex apparatus designed to produce bead slurry of several hundred ml to several liters. Said bead slurries are then incubated in the unique bioreactor that is an integral part of the Bellco device. The apparatus that is the subject of the instant disclosure is a stand-alone device designed to form droplets in small volume; it contains no bioreactor chamber suitable for cell culture.
A summary of various processes for formation of microcapsules is found in Thies, C., "Encyclopedia of the Polymer Science and Engineering", 2nd ed. (1987) 724-743, John Wiley & Sons, incorporated herein by reference. See also P. B. Deasy, Microencapsulation and Related Drug Processes, Marcel Dekker, Inc., New York, 1984.
In all of the foregoing instances, and indeed on almost any occasion in which microcapsules, beads, or other particulates are desired to be synthesized, uniform or controllable particle dimensions are desirable. In order to obtain uniform particles, the size of the droplets used as the core of the solidified particles must be controlled. The methods disclosed in the art--formation of emulsions, extrusion from needles without using a gas to control droplet size and other commonly accepted techniques, are not capable of regulating the particle size for small volumes or weights of material to be entrapped or encapsulated in a bead or microcapsule. The compact, simple, low-cost apparatus and method of this invention solves these problems and permits controlled droplet formation with small samples for a wide range of materials, organelles, and microorganisms.